Expression of gene products from genetically manipulated strains of bordetella

ABSTRACT

An expression system for expressing gene products from recombinant Bordetella strains and specific nucleic acid molecules useful in transforming Bordetella strains for such expression are described. A nucleic acid molecule may comprise a Bordetella promoter operatively coupled to a heterologous gene encoding a non-Bordetella gene product with the heterologous gene transcriptionally regulated by the Bordetella promoter. The nucleic acid molecule may further comprise a further nucleic acid molecule encoding a leader sequence for secretion of the non-Bordetella gene product. Another nucleic acid molecule may comprise a Bordetella promoter coupled to a nucleic acid sequence encoding a non-Bordetella leader sequence for secretion of a gene product, which may be a Bordetella gene product or a non-Bordetella gene product.

REFERENCE TO RELATED APPLICATION

This application is a continuation of application No. 08/393,334 filedFeb. 23, 1995 now abandoned.

FIELD TO THE INVENTION

The present invention relates to the field of molecular biology and isparticularly concerned with the expression of gene products from strainsof Bordetella.

BACKGROUND OF THE INVENTION

Bordetella pertussis, the organism responsible for whooping cough,expresses a number of virulence factors, such as pertussis toxin (PT),filamentous hemagglutinin (FHA) and pertactin (PRN). These proteins aresecreted by the organism through the use of signal peptides and/oraccessory genes (refs. 1 and 2--Throughout this specification, variousreferences are referred to in parenthesis to more fully describe thestate of the art to which this invention pertains. Full bibliographicinformation for each citation is found at the end of the specification,immediately preceding the claims. The disclosures of these referencesare hereby incorporated by reference into the present disclosure). Wehave previously demonstrated that it is possible to manipulate theexpression of these Bordetella proteins through alteration of gene copynumber (ref. 3) or the use of hybrid genes with autologous promoters(ref. 4). For example, the amount of secreted- and processed PTholotoxin was increased more than 3-fold by increasing the copy numberof the tox operon encoding PT (ref. 5). The amount of secreted andprocessed pertactin was increased 8-fold by using a hybrid gene whichreplaced the native prn promoter with the stronger fha promoter. Theyield of pertactin was further increased to 20-fold wild-type levels byadding a second copy of the hybrid gene.

Many gene products including proteins and polypeptides of commercial andmedical significance are only available in small amounts from theirnatural sources, are difficult to isolate or require modification of,for example, their primary amino acid sequence for optional use and/oractivity. Thus, many genes have been expressed by recombinant DNA meansin a variety of microbial hosts, including bacterial hosts. The geneexpressed in the microbial host is typically heterologous to the host.

Examples of bacterial hosts used for expression of heterologous proteinsinclude strains of Escherichia coli, Salmonella species (ref. 10) andBacillus subtilis (ref. 11).

Particular biological properties of strains of Bordetella make themattractive hosts for the production of certain heterologous geneproducts. Thus, many of the antigens produced by B. pertussis are large,can be multimeric and may require post-translational assembly orprocessing. For example, the pertactin antigen is produced as a 93-kDaprecursor and the mature protein is produced by excision of theN-terminal signal peptide and removal of a C-terminal fragment.Pertussis toxin is a 105 kDa exotoxin produced by B. pertussis, and isencoded by the TOX operon and consists of five polypeptide subunits (S1to S5) arranged in the typical A--B structure of bacterial toxins. TheS2, S3, S4 and S5 subunit form a pentamer (the B oligomer) which, whencombined with the S1 subunit forms the holotoxin. For PT, for example,such complex assembly cannot be achieved in E. coli (ref. 22) and, forthe 69 kd material, protein accumulated as insoluble inclusion bodies inE. coli (ref. 23). This intracellular expression in E. coli is to becontrasted with the secretion of soluble antigens by B. pertussisstrains. FHA is another large molecule (Mwt 220 kDa) secreted by B.pertussis (ref. 24).

Vibrio cholerae is the organism that causes cholera, a severe disease ofdehydration caused by diarrhoea. Many of the symptoms of cholera can beattributed to the action of cholera toxin (CT), which like B. pertussisPT, is an A/B toxin with ADP-ribosyl transferase activity. However,unlike PT which has four different B subunit components comprising apentamer, CT has a pentameric structure made up of identical subunits(ref. 6). Cholera toxin has been shown to have considerable use as amucosal adjuvant and the B subunit alone may be sufficient to generate amucosal response in some instances (ref. 7). A response is generated ifcholera toxin B (CTB) is either co-administered or chemically coupled toanother protein (ref. 8). Chimeric genes have also been engineered whichhave foreign epitopes fused to cholera toxin B and the resultant fusionproteins can sometimes induce an immune response to the foreign epitope(ref. 9).

Cholera toxin B has been expressed from recombinant V. cholerae (ref.12), E. coli (ref. 13), and S. typhimurium (ref. 14). Although B.pertussis has been used to over-express autologous proteins by genemanipulation (refs. 4 and 5), it has not heretofore been used to produceheterologous proteins.

SUMMARY OF THE INVENTION

The present invention is directed towards recombinant strains ofBordetella which express non-Bordetella gene products. Accordingly, inone aspect of the present invention, there is provided a nucleic acidmolecule comprising a Bordetella promoter operatively coupled to aheterologous gene encoding a non-Bordetella gene product, wherein theheterologous gene is transcriptionally regulated by the Bordetellapromoter.

The non-Bordetella gene product may be one of a wide variety of proteinsand polypeptides. The protein or peptide may be an enzyme, an enzymeinhibitor, an antigen, an immunogen, an allergen, a hormone, alymphokine, an immunoglobulin or fragment thereof, a toxin, a toxinsubunit, a mammalian protein, a structural protein or a receptor.

The invention is illustrated by the expression of a cholera toxinmolecule as the non-Bordetella gene product, specifically the B subunitof cholera toxin. However, any other protein or polypeptide may comprisethe expressed non-Bordetella gene product.

The Bordetella promoter employed in the nucleic acid molecule providedin accordance with this aspect of the invention may be any of theBordetella promoters, preferably the tox, prn and fha promoters from anyBordetella strain, including B. pertussis.

The heterologous gene component of the nucleic acid molecule provided inaccordance with this aspect of the invention may further comprise anucleic acid sequence encoding a leader sequence for secretion of thenon-Bordetella gene product. The leader sequence may be any sequencemediating secretion of the non-Bordetella gene product.

In one embodiment, the leader sequence is a leader sequence of aBordetella protein or subunit thereof or a fragment or analog of theBordetella protein leader sequence retaining secretion-mediatingproperties. The leader sequence may be the Bordetella pertactin leadersequence or a pertussis toxin subunit leader sequence, such as that forthe S1 subunit, of any Bordetella strain, including B. pertussis.

Alternatively, in another embodiment, the leader sequence is a leadersequence of a non-Bordetella protein or subunit thereof or a fragment oranalog of the non-Bordetella protein leader sequence retainingsecretion-mediating properties. The non-Bordetella gene product may be asecreted gene product, in which case the non-Bordetella leader sequencepreferably is the native leader sequence of the secreted gene product.

In an illustrative example of the latter embodiment of the invention,the secreted gene product may be a cholera toxin molecule, for example,the B subunit thereof and the leader sequence may be the cholera toxin Bsubunit leader sequence.

Specific combinations of promoter, leader sequence and heterologous geneproduct sequence are provided herein, including those designedtoxp/CTB-L/ctb, fhap/CTB-L/ctb, toxp/S1-L/ctb, toxp/PRN-L/ctb,fhap/S1-L/ctb and fhap/PRN-L/ctb.

The nucleic acid molecule provided in accordance with this aspect of theinvention may further comprise a first DNA sequence corresponding to a5' flanking sequence of a selected Bordetella gene and disposed at the5' end of the nucleic acid molecule and a second DNA sequencecorresponding to a 3' flanking sequence of the selected Rordetella geneand disposed at the 3' end of the nucleic acid molecule. The first andsecond DNA sequences permit specific integration of the nucleic acidmolecule into the genome of a Bordetella species, preferably B.pertussis, at a locus corresponding to the selected Bordetella gene. TheBordetella promoter present in the nucleic acid molecule may be that ofthe selected Bordetella gene providing the flanking sequences. Theselected Bordetella gene may be any of the Bordetella genes, includingthe tox, prn or fha gene of a Bordetella strain, preferably B.pertussis.

The nucleic acid molecule with flanking regions as described above maybe provided in a plasmid adapted for transformation of a Bordetellastrain, preferably a B. pertussis strain. Specific plasmids have beenprepared herein, as described in more detail below and are identified asplasmids DS-546-1, JB-898-2-1, DS-729-1-1, DS-729-2-1, JB-1201-4 andJB-1141-5.

Another aspect of the invention provides a recombinant strain ofBordetella, which may be a B. pertussis strain, a B. parapertussisstrain, a B. bronchiseptica strain or a B. avium strain, particularly aB. pertussis strain, containing the nucleic acid molecule provided inthe above-described aspect of the invention, integrated into the genomethereof and expressing the non-Bordetella gene product. One specificrecombinant B. pertussis strain provided herein is B. pertussis strain694-46, which has been deposited with the American Type CultureCollection, Rockville, Md., USA, on Jan. 11, 1995 under the terms of theBudapest Treaty as ATCC Accession number 55,654. The non-Bordetella geneproduct may be obtained by culturing a recombinant strain providedherein.

In a further aspect of the invention, there is provided a nucleic acidmolecule comprising a Bordetella promoter coupled to a nucleic acidsequence encoding a non-Bordetella leader sequence for secretion of agene product. The secreted gene product may be a Bordetella gene productor a non-Bordetella gene product.

The Bordetella promoter component of the nucleic acid molecule providedin accordance with this further aspect of the invention may be any ofthe Bordetella promoters, preferably the tox, prn or fha promoter of aBordetella strain, preferably B. pertussis.

The non-Bordetella leader sequence component of the nucleic acidmolecule provided in accordance with this further aspect of theinvention may be any of the leader sequences mediating secretion of agene product, including bacterial (prokaryotic) leader sequences (suchas E. coli leader sequences including rlpB, pal ompA, pilin gene leadersequences and H. influenzae leader sequences including the transferrinreceptor protein leader sequence), eukaryotic leader sequences(including mammalian) and viral leader sequences. Some appropriateleader sequences for use in aspects of the present invention aredescribed in reference 25. In an illustrative example of this furtheraspect of the invention, the non-Bordetella leader sequence may be thatfor the cholera toxin B subunit.

A recombinant strain of Bordetella, such as a B. pertussis strain, a B.parapertussis strain, B. bronchiseptica strain or a B. avium strain,preferably a B. pertussis strain, may contain the nucleic acid moleculeprovided in accordance with this further aspect of the invention andsecrete the gene product. Such recombinant strain of Bordetella may becultured under a range of appropriate conditions to secrete the geneproduct.

In an additional aspect of the invention, the heterologous gene may beprovided with optimized codons for Bordetella expression. One example ofsuch heterologous gene has the nucleic acid sequence of FIG. 1, whichencodes the B subunit of cholera toxin and constitutes an embodiment ofthis additional aspect of the invention.

The present invention, therefore, provides an expression system forexpressing gene products from recombinant Bordetella strains andspecific nucleic acid molecules useful in transforming Bordetellastrains for such expression. The expressed gene products have a varietyof uses, depending on the form and nature of the product produced, aswill be evident to a person skilled in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be further understood from the followingdetailed description and examples with reference to the accompanyingdrawings, in which:

FIG. 1 shows the sequence of a synthetic cholera toxin B subunit gene(SEQ ID NO: 1) and its derived amino acid sequence (SEQ ID NO: 2), basedupon strain 569B of V. cholerae;

FIG. 2 shows the construction scheme for plasmid DS-546-1 which containsthe toxp/CTB-L/ctb gene;

FIG. 3 shows the sequences of oligonucleotides (SEQ ID NOS: 3 to 16)used for the construction of plasmid DS-546-1;

FIG. 4 shows the construction scheme for plasmid JB-898-2-1 whichcontains the fhap/CTB-L/ctb gene;

FIG. 5 shows the sequences of oligonucleotides (SEQ ID NOS: 17 to 20)used to construct plasmid JB-898-2-1;

FIG. 6 shows the construction scheme for plasmid DS-729-1-1 whichcontains the toxp/S1-L/ctb gene;

FIG. 7 shows the sequences of oligonucleotides (SEQ ID NOS: 21 to 30)used to construct plasmid DS-729-1-1;

FIG. 8 shows the construction scheme for plasmid DS-729-2-1 whichcontains the toxp/PRN-L/ctb gene;

FIG. 9 shows the sequences of oligonucleotides (SEQ ID NOS: 31 to 40)used to construct plasmid DS-729-2-1;

FIG. 10 shows the construction scheme for plasmid JB-1201-4 whichcontains the fhap/S1-L/ctb gene;

FIG. 11 shows the sequences of oligonucleotides (SEQ ID NOS: 41 to 48)used to construct plasmid JB-1201-4;

FIG. 12 shows the construction scheme for plasmid JB-1141-5 whichcontains the fhap/PRN-L/ctb gene;

FIG. 13 shows the sequences of oligonucleotides (SEQ ID NOS: 49 to 56)used to construct plasmid JB-1141-5;

FIG. 14 shows the chromosomal maps of the genes at the fha and tox lociand the corresponding Southern blot showing the correct chromosomalintegration. Chromosomal DNA was digested with Bgl II and hybridizedwith the approximately 300 bp ctb probe indicated in the figure. Lane 1,strain 694-46 (fhap/PRN-L/ctb); lane 2, strain 694-54 (fhap/S1-L/ctb);lane 3, strain 694-12 (toxp/PRN-L/ctb); lane 4, strain 694-4(toxp/S1-L/ctb); lane 5, strain 10536 (wild-type B. pertussis); and

FIG. 15 shows the SDS PAGE and corresponding Western blot of recombinantB. pertussis strain 694-46 which expresses CTB. Lane 1, acetoneprecipitated supernatant from strain 694-46 (fhap/PRN-L/ctb), boiled inSDS; lane 2, acetone precipitated supernatant from strain 694-46,unboiled; lane 3, cell pellet from strain 694-46, boiled in SDS; lane 4,cell pellet from strain 694-46, unboiled; lane 5, acetone precipitatedsupernatant from strain 10536 (wild-type B. pertussis); lane 6, cellpellet from strain 10536; lane 7, purified cholera toxin (Sigma), boiledin SDS; lane 8, purified cholera toxin, unboiled.

In some of the above figures, the following abbreviations are used:

toxp is the B. pertussis tox promoter

fhap is the B. pertussis fha promoter

ctb is the synthetic cholera toxin B gene (SEQ ID NO: 1)

CTB-L is the sequence encoding the cholera toxin B subunit leadersequence

S1-L is the sequence encoding the pertussis toxin subunit S1 leadersequence

PRN-L is the sequence encoding the pertussis pertactin leader sequence

Restriction enzyme recognition sites are B, BamH I; Bg, Bgl II; H, HindIII; K, Kpn I; R, EcoR I; S, Sac I; and Hf, HinfI

CAP is calf alkaline phosphatase

GENERAL DESCRIPTION OF THE INVENTION

Bordetella pertussis 10536 is the vaccine production strain of theassignee hereof and it has been used as the initial strain for all thework detailed by the inventors herein. The genes for B. pertussis PT,FHA and pertactin and V. cholerae CT have been cloned and sequenced(Refs. 15 to 19) and the promoter regions and transcriptional starts ofthe structural genes have been determined.

The inventors have generated hybrid genes by substituting the B.pertussis native structural genes encoding a leader sequence and/ormature protein by gene segments encoding native, autologous, orheterologous leader peptides and a mature foreign protein. This wasaccomplished by fusing the promoters with the gene segment encoding theleader peptide at the ATG start codon, followed by the structural genefor the mature foreign protein joined at the natural cleavage site ofthe signal sequence. Such fusions result in a native promoter, a native,autologous, or heterologous leader peptide, and a heterologousstructural gene. The resultant hybrid genes then have been integrated byhomologous recombination into the chromosome of B. pertussis at the locicorresponding to the gene from which the promoters were derived.

As examples of the use of hybrid genes expressing foreign proteins,genes have been created containing a tox promoter with the cholera toxinB leader peptide and mature cholera toxin B sequence; a tox promoterwith the pertussis toxin subunit S1 leader peptide and mature choleratoxin B sequence; a tox promoter with the pertactin leader peptide andmature cholera toxin B sequence; an fha promoter with the cholera toxinB leader peptide and mature cholera toxin B sequence; an fha promoterwith the pertussis toxin subunit S1 leader peptide and mature choleratoxin B sequence; and an fha promoter with the pertactin leader peptideand mature cholera toxin B sequence. A number of B. pertussis strainshave been generated to demonstrate the success of this strategy.

The efficiency of expression of the foreign CTB protein is dependentupon both the promoter and the leader peptide which precede thestructural ctb gene. The use of the fha promoter in the hybrid genesresults in a higher level of expression of the foreign protein than whenthe tox promoter is used. This phenomenon was also observed when hybridgenes were used to express autologous proteins from B. pertussis (ref.4). For the leader peptides, expression levels varied as follows:pertactin>PT subunit S1>cholera toxin B. The best combination ofpromoter and leader peptide in the hybrid genes expressing foreignproteins was the fha promoter with the pertactin leader peptide.

The CTB expressed by the recombinant B. pertussis strains is producedand is a pentamer as demonstrated by SDS PAGE and Western blot analysisof unboiled (pentameric) and boiled (monomeric) samples. The CTB bindsto GM1 as demonstrated by ELISA. Thus, a complex foreign protein whichhas authentic structure and binding functions can be secreted by therecombinant Bordetella strains of the invention.

It has been clearly demonstrated that the structural gene of a foreignprotein may be fused to a B. pertussis promoter through a gene fragmentencoding a native, autologous, or heterologous leader peptide to expressforeign proteins.

BIOLOGICAL DEPOSITS

B. pertussis strain 694-46 which contains the fhap/PRN-L/ctb hybrid geneat the fha locus has been deposited with the American Type CultureCollection (ATCC) located at Rockville, Md., USA, pursuant to theBudapeast Treaty and prior to the filing of this application. The ATCCaccess number is 55,654.

Samples of the deposited strain will become available to the public uponthe grant of a patent based on this United States patent application.The invention described and claimed herein is not limited in scope bythe strain deposited, since the deposited embodiment is intended only asan illustration of the invention. Any equivalent or similar strains tothat deposited are within the scope of the invention.

EXAMPLES

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific Examples. These Examples are described solely for the purposesof illustration and are not intended to limit the scope of theinvention. Changes in form and substitution of equivalents arecontemplated as circumstances may suggest or render expedient. Althoughspecific terms have been employed herein, such terms are intended in adescriptive sense and not for purposes of limitations.

Methods of molecular genetics, protein biochemistry, and immunology usedbut not explicitly described in this disclosure and these Examples areamply within the ability of those skilled in the art.

Example 1

This Example illustrates the construction of a synthetic gene encodingthe cholera toxin B subunit.

A gene encoding the cholera toxin B subunit was synthesized from anumber of oligonucleotides. The oligonucleotides were synthesized on anABI model 380B DNA synthesizer and purified by gel electrophoresis.Nucleotide sequences were confirmed by automated DNA sequencing on theABI model 370A DNA sequencer using dye terminator chemistry. Forconstruction of the synthetic gene encoding cholera toxin subunit,codons preferred by B. pertussis were selected. The nucleotide sequenceof the hybrid gene is shown in FIG. 1 (SEQ ID NO: 1).

Example 2

This Example illustrates the construction of plasmid DS-546-1 containingthe toxp/CTB-L/ctb gene.

Oligonucleotides 2769.SL (SEQ ID NO: 3), 2770.SL (SEQ ID NO: 4), 2771.SL(SEQ ID NO: 5), 2780.SL (SEQ ID NO: 14), 2781.SL (SEQ ID NO: 15) and2782.SL (SEQ ID NO: 16) (FIG. 3) contain part of the tox promoter,encode the cholera toxin B leader peptide, and contain ˜70 bp of the5'-end of the ctb gene encoding the mature cholera toxin B subunitprotein. Plasmid S-3616-2 is an 8.6 kb pBR322-based plasmid containing2.5 kb of the 5'- and 1.3 kb of the 3'-flanking regions for the fhastructural gene between Bgl II and Kpn I sites (FIG. 2).Oligonucleotides 2769.SL, 2770.SL, 2771.SL, 2780.SL, 2781.SL, and2782.SL were kinased, annealed, and ligated with the 4.8 kb vectorfragment of S-3616-2 which had been digested with Bgl II and Kpn I toform plasmid JB-867-1-1, which contains a toxp/CTB-L/5'ctb gene inserton a ˜220 bp Kpn I/Bgl II fragment.

Oligonucleotides 2772.SL to 2779.SL (SEQ ID NOs: 6 to 13) (see FIG. 3)encode the remaining ˜250 bp of the ctb gene. They were kinased,annealed, and ligated with the 6.1 kb vector fragment from plasmidS-3616-2, which had been digested with Bgl II and EcoR I, to generateplasmid DS-525-1-1.

The Kpn I/Bgl II fragment from plasmid JB-867-1-1 and the Bgl II/EcoR Ifragment of plasmid DS-525-1-1 were ligated into pUC18, which had beendigested with Kpn I and EcoR I, to generate plasmid DS-534-1 which thuscontains the entire toxp/CTB-L/ctb gene.

Plasmid S-3484-3-27 is a 14.2 kb pUC-based plasmid containing a mutanttox gene between the 5'- and 3'-tox flanking regions. Digestion with KpnI and BamH I excised ˜4.7 kb of the tox structural gene. The ˜470 bp KpnI/BamH I hybrid gene fragment from DS-534-1 was ligated into the KpnI/BamH I vector fragment from S-3484-3-27 to generate plasmid DS-546-1(FIG. 2) which contains the toxp/CTB-L/ctb hybrid gene between the toxflanking regions. This plasmid was used for insertion of thetoxp/CTB-L/ctb gene into the tox locus of B. pertussis, generatingstrain 492-320.

Example 3

This Example describes the construction of plasmid JB-898-2-1 containingthe fhap/CTB-L/ctb gene.

Plasmid S-3658-1 contains a ˜210 bp EcoR I/Hinf I fragment of the fhapromoter (FIG. 4). Oligonucleotides 2821.SL to 2824.SL (SEQ ID NOS: 17to 20) (see FIG. 5) encode the remaining 36 bp of the fha promoter andencode most of the cholera toxin B leader peptide. Oligonucleotides2821.SL to 2824.SL were kinased, annealed, and ligated with the EcoRI/Hinf I fha promoter fragment from S-3658-1 into pUC18 which had beendigested with EcoR I and Sac I. Plasmid JB-881-2 thus contains a portionof the fhap/CTB-L hybrid gene on a 290 bp EcoR I/Sac I fragment.Digestion of plasmid DS-534-1, which contains the completetoxp/CTB-L/ctb gene, with EcoR I and Sac I excised a ˜340 bp fragment ofthe ctb gene. Ligation of the EcoR I/Sac I fragments from DS-534-1 andJB-881-2 into S-3616-2, which had been digested with EcoR I anddephosphorylated, generated plasmid JB-898-2-1 (FIG. 4) which containsthe entire fhap/CTB-L/ctb gene between the fha flanking regions. Thisplasmid was used to insert the fhap/CTB-L/ctb hybrid gene into the fhalocus of B. pertussis, generating strain 492-363.

Example 4

This Example illustrates the construction of plasmid DS-729-1-1 whichcontains the toxp/S1-L/ctb hybrid gene.

Plasmid DS-534-1 contains the complete toxp/CTB-L/ctb gene on a ˜470 bpKpn I/EcoR I fragment (FIG. 6). Oligonucleotides 2769.SL (SEQ ID NO:21), 3220.SL (SEQ ID NO: 22), 3221.SL (SEQ ID NO: 23), 3222.SL (SEQ IDNO: 24), 3212.SL (SEQ ID NO: 25), 2782.SL (SEQ ID NO: 30), 3225.SL (SEQID NO: 29), 3224.SL (SEQ ID NO: 28), 3223.SL (SEQ ID NO: 27) and 3213.SL(SEQ ID NO: 26) (see FIG. 7) contain part of the tox promoter, encodethe pertussis toxin subunit S1 leader peptide, and contain ˜70 bp of the5'-end of ctb encoding the mature cholera toxin B subunit protein. Theoligonucleotides were kinased, annealed, and ligated with the 3 kb KpnI/Bgl II vector fragment from DS-534-1 (FIG. 6) to generate plasmidDS-700-2-1 which thus contains the complete toxp/S1-L/ctb gene on a ˜505bp Kpn I/BamH I fragment. The Kpn I/BamH I tox structural gene wasexcised from plasmid S-3484-3-27 and the toxp/S1-L/ctb gene inserted, togenerate plasmid DS-729-1-1 which contains the toxp/S1-L/ctb genebetween the tox flanking regions (FIG. 6). This plasmid was used toinsert the toxp/S1-L/ctb hybrid gene into the tox locus of B. pertussis,generating strain 694-4.

Example 5

This Example illustrates the construction of plasmid DS-729-2-1 whichcontains the toxp/PRN-L/ctb hybrid gene.

Oligonucleotides 2769.SL (SEQ ID NO: 31), 3210.SL (SEQ ID NO: 32),3217.SL (SEQ ID NO: 33), 3211.SL (SEQ ID NO: 34), 3212.SL (SEQ ID NO:35), 3213.SL (SEQ ID NO: 36), 3214.SL (SEQ ID NO: 37), 3215.SL (SEQ IDNO: 38), 3218.SL (SEQ ID NO: 39) and 2782.SL (SEQ ID NO: 40) (see FIG.9) contain part of the tox promoter, encode the pertactin leaderpeptide, and contain ˜70 bp of the ctb gene encoding the mature choleratoxin B subunit protein. The oligonucleotides were kinased, annealed,and ligated into DS-534-1 (FIG. 8) which had been digested with KpnI andBgl II to delete ˜220 bp of the 5'-end of the toxp/CTB-L/ctb hybrid gene(FIG. 5A). Plasmid DS-707-6 contains the complete toxp/PRN-L/ctb hybridgene on a ˜505 bp Kpn I/BamH I fragment. S-3484-3-27 was digested withKpn I and BamH I to excise the tox structural gene and the hybrid genewas inserted. The resulting plasmid DS-729-2-1 contains thetoxp/PRN-L/ctb gene between the tox flanking regions (FIG. 8) and wasused for insertion of the hybrid gene into the tox locus of B.pertussis, generating strain 694-12.

Example 6

This Example illustrates the construction of plasmid JB-1201-4 whichcontains the fhap/S1-L/ctb hybrid gene.

Plasmid S-3658-1 contains a ˜210 bp EcoR I/Hinf I fragment of the fhapromoter (FIG. 10). Oligonucleotides 3226.SL (SEQ ID NO: 41), 3221.SL(SEQ ID NO: 42), 3222.SL (SEQ ID NO: 43), 3212.SL (SEQ ID NO: 44),3213.SL (SEQ ID NO: 45), 3223.SL (SEQ ID NO: 46), 3224.SL (SEQ ID NO:47) and 3227.SL (SEQ ID NO: 48) (FIG. 11) contain part of the fhapromoter, encode the pertussis toxin subunit S1 leader peptide, andcontain the first ˜70 bp of the ctb gene encoding the mature choleratoxin B subunit protein. Plasmid pUC8/BgXb is a pUC8 derived plasmidwith extra restriction enzyme sites for Bgl II and Xba I in the multiplecloning site (FIG. 10). The oligonucleotides were kinased, annealed, andligated with the EcoR I/Hinf I fha promoter fragment into pUC8 /BgXbwhich had been digested with EcoR I and Bgl II. Plasmid JB-1190-1 thuscontains the fhap/S1-L/5'ctb hybrid gene on a ˜414 bp EcoR I/Bgl IIfragment. Plasmid DS-534-1 was digested with Bgl II and EcoR I to excisethe ˜250 bp 3'-ctb fragment, which was ligated with the EcoR I/Bgl IIhybrid gene fragment into S-3616-2, which had been digested with EcoR Iand dephosphorylated. The resulting plasmid JB-1201-4 thus contains thecomplete fhap/S1-L/ctb hybrid gene between the fha flanking regions, ina reverse orientation with respect to the flanking regions (FIG. 10).This plasmid was used to introduce the fhap/S1-L/ctb hybrid gene intothe fha locus of B. pertussis, generating strain 694-54.

Example 7

This Example illustrates the construction of plasmid JB-1141-5 whichcontains the fhap/PRN-L/ctb hybrid gene.

Oligonucleotides 3216.SL (SEQ ID NO: 49), 3217.SL (SEQ ID NO: 50),3211.SL (SEQ ID NO: 51), 3212.SL (SEQ ID NO: 52), 3213.SL (SEQ ID NO:53), 3214.SL (SEQ ID NO: 54), 3215.SL (SEQ ID NO: 55) and 3219.SL (SEQID NO: 56) contain part of the fha promoter, encode the pertactin leaderpeptide, and contain the first ˜70 bp of the ctb gene encoding themature cholera toxin B subunit protein (FIG. 13). The oligonucleotideswere kinased, annealed, and ligated with the 210 bp EcoR/Hinf I fhapromoter fragment from S-3658-1 (FIG. 12), into pUC8 /BgXb which hadbeen digested with EcoR I and Bgl II. Plasmid JB-1076-1-2 contains thefhap/PRN-L/5'ctb hybrid gene on a ˜414 bp EcoR I/Bgl II fragment and wasligated with the remainder of the ctb gene, excised from DS-534-1 on a˜250 bp Bgl II/EcoR I fragment, into the S-3616-2 vector which had beendigested with EcoR I and dephosphorylated. The resulting plasmidJB-1141-5 (FIG. 12) thus contains the complete fhap/PRN-L/ctb genebetween the fha flanking regions and was used to insert the hybrid geneinto the fha locus of B. pertussis, generating strain 694-46.

Example 8

This Example illustrates the generation of recombinant B. pertussisstrains.

B. pertussis strain 29-8 (ATCC 53,973) is a tox-deleted derivative ofstrain 10536 and is described in ref. 20. This strain was used as thehost strain for genomic introduction of plasmids DS-546-1(toxp/CTB-L/ctb), DS-729-1-1 (toxp/S1-L/ctb) and DS-729-2-1(toxp/PRN-L/ctb). B. pertussis strain 590-508 is an fha- and tox-deletedderivative of strain 10536 and was used to genomically introduceplasmids JB-898-2-1 (fhap/CTB-L/ctb), JJB-1201-4 (fhap/S1-L/ctb), andJB-1141-5 (fhap/PRN-L/ctb). Plasmids were introduced into the B.pertussis strains by electroporation as described by Zealey et al. (ref.5 and 20). A single cross-over event resulted in integration of theentire plasmid generating str^(S) Ap^(R) ctb⁺ strains. Selecting onstreptomycin (100 μg ml⁻¹) resulted in a second homologous recombinationevent which deletes all plasmid sequences and generated strains 694-4,694-12, 694-46, and 694-54 respectively. Strain 694-46 has beendeposited with ATCC (ATCC 55,654). The second cross-over did not occurfor strains 492-320 and 492-363, thus these strains include the entireplasmids DS-546-1 and JB-898-2-1 respectively.

The identification of the recombinant B. pertussis strains obtained andtheir derivations are summarized in Table 1 below.

Example 9

This Example illustrates a Southern blot analysis of recombinant B.pertussis strains to confirm the correct chromosomal location of theintegrated genes.

Chromosomal DNA was isolated from B. pertussis strains 10536, 694-4,694-12, 694-46, and 694-54, digested with restriction enzyme Bgl II,electrophoresed, and transferred to Gene Screen Plus (Dupont). The blotwas hybridized with a ˜300 bp Bgl II/BamH I ctb-specific probe and theresults are shown in FIG. 14. The appearance of a ctb-specific 1.5 kbBgl II fragment confirmed the correct location of integration of thetoxp/S1-L/ctb and toxp/PRN-L/ctb genes at the tox locus of B. pertussisstrains 694-4 and 694-12 respectively. The appearance of a specific 11kb Bgl II fragment confirmed the correct location of the fhap/S1-L/ctband fhap/PRN-L/ctb hybrid genes at the fha locus in B. pertussis strains694-54 and 694-46 respectively.

Example 10

This Example illustrates the growth of recombinant B. pertussis strainsand expression of cholera toxin B subunit.

Recombinant B. pertussis strains were grown in modified Stainer-Scholtemedium containing 0.2% heptakis(2,4-O-dimethyl)β-cyclodextrin (ref. 21)in 10 ml culture. 1 ml of culture was centrifuged at 12,000×g for 4 minand the resulting supernatant was analysed by ELISA, SDS PAGE, and/orWestern blot. The cell pellets were resuspended in 1 ml of media andlysed by sonication at 25μ for 45 sec, on ice with a Soniprep model 150sonicator. Then, the cell debris was removed by centrifugation at12000×g for 4 min, and the supernatant removed and analysed by ELISA.

The level of expression of cholera toxin B subunit from various B.pertussis strains is shown in Table 2 below.

Example 11

This Example illustrates the ELISA assay used for the quantitation ofcholera toxin B expression.

Nunc Maxisorp 96 well plates were coated with monosialoganglioside--GM1(Sigma) and incubated overnight at room temperature. The plates werewashed and blocked with PBS containing 0.1% BSA for 45 min at roomtemperature. Two-fold serial dilutions of samples were added and theplates incubated for 1 h at room temperature. Cholera toxin B (ListBiologicals #103C), growth media, wild-type B. pertussis supernatant andcell pellets were used as standard and controls. CTB was detected usinggoat anti-choleragenoid (anti-CTB, List Biologicals #GAC-01C) as primaryantibody and HRP-conjugated affinity purified rabbit anti-goat IgG assecondary antibody.

Example 12

This Example illustrates the analysis of the expressed recombinantproteins by SDS-PAGE and Western blot.

Culture supernatants were acetone-precipitated and resuspended in 0.1volume of SDS lysis buffer. An equal volume of 2×Laemmli sample bufferwas added and the samples were either resolved directly on a 17.5% SDSPAGE gel or boiled for 5 min, before being resolved to convert the CTBpentamer to the monomeric form. Cell pellets were resuspended in 0.1volume of SDS lysis buffer, an equal volume of 2×Laemmli sample bufferadded, and the samples treated as above.

For Western blot analysis, proteins were transferred onto Gene ScreenPlus nylon membranes (Dupont) and probed with goat anti-choleragenoidIgG antibody (anti-CTB, List Biologicals #GAC-01C). Detection wasperformed with alkaline phosphatase-conjugated donkey anti-goat IgG,using dig chemiluminescence (Boehringer Mannheim). A cholera toxinstandard (Sigma) contained both CTA and CTB.

FIG. 9 shows the expression of CTB as a pentamer in culture supernatantsof strain 694-46 (fhap/PRN-L/ctb). The identity of the cholera toxin isindicated by the correct molecular weight; specific-reactivity withanti-CTB antiserum and the conversion of the pentameric form of choleratoxin B subunit protein to the monomeric form by boiling.

SUMMARY OF THE DISCLOSURE

In summary of this disclosure, the present invention provides a novelgene product expression method having particular application toBordetella species. Modifications are possible within the scope of thisinvention.

                  TABLE 1                                                         ______________________________________                                        Recombinant B. pertussis strains                                                  Strain   Genotype    Genetic Locus                                                                            Plasmid Used                              ______________________________________                                        492-320  toxp/CTB-L/ctb                                                                            tox          DS-546-1                                      492-363 fhap/CTB-L/ctb fha JB-898-2-1                                         694-4 toxp/S1-L/ctb tox DS-729-1-1                                            694-12 toxp/PRN-L/ctb tox DS-729-2-1                                          694-54 fhap/S1-L/ctb fha JB-1201-4                                            694-46 fhap/PRN-L/ctb fha JB-1141-5                                         ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        Cholera toxin B expression from recombinant B. pertussis strains                                   CTB Production (ng/ml)                                   Plasmid Strain  promoter leader                                                                              secreted                                                                              internal                               ______________________________________                                        DS-546-1                                                                              492-320 tox      CTB   2.7     0                                        DS-729-1-1 694-4 tox S1 1 ± 0.5 9.7 ± 12.9                              DS-729-2-1 694-12 tox PRN 3.3 ± 7.3 44.9 ± 71.2                         JB-898-2-1 492-363 fha CTB 5.4 9.0                                            JB-1201-4 694-54 fha S1 116.4 ± 41.3 59.7 ± 14.1                        JB-1141-5 694-46 fha PRN 11,287 ± 5,509.8 ±                                 3,029.7 785.4                                                           ______________________________________                                    

REFERENCES

1. Johnson and Burns. 1994. J. Bacteriol. 176: 5350.

2. Locht et al. 1992. EMBO J. 11: 3175.

3. EP patent application 523 976.

4. EP patent application 453 216.

5. Zealey et al. 1992. Appl. Environ. Microbiol. 58: 208.

6. Burnette. 1994. Structure 2: 151.

7. Holmgren et al. 1993. Vaccine 11: 1179.

8. Wu and Russell. 1994. Vaccine 12: 215.

9. Dertzbough and Elson. 1993. Infect. Immun. 61: 384.

10. Cardenas and Clement. 1993. Vaccine 11: 126.

11. Airaksinen et al. 1991. Biotechnology Lett 13: 305.

12. Lebens et al. 1993. Bio/Technology 11: 1574.

13. Burnette et al. 1991. Infect. Immun. 59: 4266.

14. Klauser et al. 1990. EMBO J. 1991.

15. Nicosia et al. 1986. Proc. Natl. Acad. Sci. (USA) 83: 4631.

16. Loosmore et al. 1989. Nucleic Acid. Res. 17: 8365.

17. Relman et al. 1989. Proc. Natl. Acad. Sci. (USA) 86: 2637.

18. Charles et al. 1989. Proc. Natl. Acad. Sci. (USA) 86: 3554.

19. Dams et al. 1991. Biochen. Biophys. Acta. 1090: 139.

20. Zealey et al. 1990. Bio/Technology 8: 1025.

21. Imaizumi et al. 1983. Infect. Immun. 41: 1138.

22. Nicosia et al. 1987. Infect. Immun. 55: 963.

23. Makoff et al. 1990. Bio/Technology 8: 1030.

24. Domenighimi et al. 1990. Molec. Microbiol. 4: 787.

25. Watson, M. 1984. Nucl. Acids. Res. 12: 5145.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 56                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 312 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..309                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - ACC CCG CAG AAC ATC ACC GAC CTG TGC GCC GA - #A TAC CAC AAC ACC        CAG       48                                                                    Thr Pro Gln Asn Ile Thr Asp Leu Cys Ala Gl - #u Tyr His Asn Thr Gln            1               5 - #                 10 - #                 15              - - ATC CAT ACC CTG AAC GAC AAG ATC TTC AGC TA - #C ACC GAA AGC CTG GCC           96                                                                       Ile His Thr Leu Asn Asp Lys Ile Phe Ser Ty - #r Thr Glu Ser Leu Ala                        20     - #             25     - #             30                  - - GGC AAG CGC GAA ATG GCC ATC ATC ACC TTC AA - #G AAC GGC GCC ACC TTC          144                                                                       Gly Lys Arg Glu Met Ala Ile Ile Thr Phe Ly - #s Asn Gly Ala Thr Phe                    35         - #         40         - #         45                      - - CAG GTC GAA GTC CCG GGC AGC CAG CAT ATC GA - #C AGC CAG AAG AAG GCC          192                                                                       Gln Val Glu Val Pro Gly Ser Gln His Ile As - #p Ser Gln Lys Lys Ala                50             - #     55             - #     60                          - - ATC GAA CGC ATG AAG GAC ACC CTG CGC ATC GC - #C TAC CTG ACC GAA GCC          240                                                                       Ile Glu Arg Met Lys Asp Thr Leu Arg Ile Al - #a Tyr Leu Thr Glu Ala            65                 - # 70                 - # 75                 - # 80       - - AAG GTC GAA AAG CTG TGC GTC TGG AAC AAC AA - #G ACC CCG CAT GCC ATC          288                                                                       Lys Val Glu Lys Leu Cys Val Trp Asn Asn Ly - #s Thr Pro His Ala Ile                            85 - #                 90 - #                 95              - - GCC GCC ATC AGC ATG GCC AAC TAA     - #                  - #                   312                                                                     Ala Ala Ile Ser Met Ala Asn                                                               100                                                                - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 103 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Thr Pro Gln Asn Ile Thr Asp Leu Cys Ala Gl - #u Tyr His Asn Thr Gln        1               5 - #                 10 - #                 15              - - Ile His Thr Leu Asn Asp Lys Ile Phe Ser Ty - #r Thr Glu Ser Leu Ala                   20     - #             25     - #             30                  - - Gly Lys Arg Glu Met Ala Ile Ile Thr Phe Ly - #s Asn Gly Ala Thr Phe               35         - #         40         - #         45                      - - Gln Val Glu Val Pro Gly Ser Gln His Ile As - #p Ser Gln Lys Lys Ala           50             - #     55             - #     60                          - - Ile Glu Arg Met Lys Asp Thr Leu Arg Ile Al - #a Tyr Leu Thr Glu Ala       65                 - # 70                 - # 75                 - # 80       - - Lys Val Glu Lys Leu Cys Val Trp Asn Asn Ly - #s Thr Pro His Ala Ile                       85 - #                 90 - #                 95              - - Ala Ala Ile Ser Met Ala Asn                                                          100                                                                - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 75 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - CGGTCACCGT CCGGACCGTG CTGACCCCCC TGCCATGGTG TGATCCGTAA AA -             #TAGGCACC     60                                                                 - - ATCAAAACGC AGAGG              - #                  - #                      - #    75                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 68 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - GGAAGACGGG ATGATCAAGA TCAAGTTCGG CGTCTTCTTC ACCGTCCTGC TG -             #AGCTCCGC     60                                                                 - - CTACGCCC                - #                  - #                       - #          68                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 73 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - ATGGCACCCC GCAGAACATC ACCGACCTGT GCGCCGAATA CCACAACACC CA -            #GATCCATA     60                                                                 - - CCCTGAACGA CAA              - #                  - #                      - #      73                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 63 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - GATCTTCAGC TACACCGAAA GCCTGGCCGG CAAGCGCGAA ATGGCCATCA TC -             #ACCTTCAA     60                                                                 - - GAA                  - #                  - #                  - #                 63                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 57 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - CGGCGCCACC TTCCAGGTCG AAGTCCCGGG CAGCCAGCAT ATCGACAGCC AG - #AAGAA            57                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 63 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - GGCCATCGAA CGCATGAAGG ACACCCTGCG CATCGCCTAC CTGACCGAAG CC -             #AAGGTCGA     60                                                                 - - AAA                  - #                  - #                  - #                 63                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 68 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - GCTGTGCGTC TGGAACAACA AGACCCCGCA TGCCATCGCC GCCATCAGCA TG -             #GCCAACTA     60                                                                 - - AGGATCCG                - #                  - #                       - #          68                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 62 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - AATTCGGATC CTTAGTTGGC CATGCTGATG GCGGCGATGG CATGCGGGGT CT -            #TGTTGTTC     60                                                                 - - CA                  - #                  - #                  - #                  62                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 65 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - GACGCACAGC TTTTCGACCT TGGCTTCGGT CAGGTAGGCG ATGCGCAGGG TG -             #TCCTTCAT     60                                                                 - - GCGTT                 - #                  - #                  -      #            65                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 55 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - CGATGGCCTT CTTCTGGCTG TCGATATGCT GGCTGCCCGG GACTTCGACC TG - #GAA              55                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 69 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - - GGTGGCGCCG TTCTTGAAGG TGATGATGGC CATTTCGCGC TTGCCGGCCA GG -             #CTTTCGGT     60                                                                 - - GTAGCTGAA                - #                  - #                      - #         69                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 87 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - - GATCTTGTCG TTCAGGGTAT GGATCTGGGT GTTGTGGTAT TCGGCGCACA GG -             #TCGGTGAT     60                                                                 - - GTTCTGCGGG GTGCCATGGG CGTAGGC          - #                  - #                 87                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 66 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                              - - GGAGCTCAGC AGGACGGTGA AGAAGACGCC GAACTTGATC TTGATCATCC CG -             #TCTTCCCC     60                                                                 - - TCTGCG                 - #                  - #                  -     #           66                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                              - - TTTTGATGGT GCCTATTTTA CGGATCACAC CATGGCAGGG GGGTCAGCAC GG -             #TCCGGACG     60                                                                 - - GTGACCGGTA C               - #                  - #                      - #       71                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 46 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                              - - ATTCTGCCGA TTACTTCACT TCGCTGGTCG GAATATGATC AAGATC   - #                     46                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                              - - AAGTTCGGCG TCTTCTTCAC CGTCCTGCTG AGCT       - #                  -      #        34                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 40 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                              - - CAGCAGGACG GTGAAGAAGA CGCCGAACTT GATCTTGATC     - #                      - #    40                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                              - - ATATTCCGAC CAGCGAAGTG AAGTAATCGG CAG       - #                  - #             33                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:21:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 75 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                              - - CGGTCACCGT CCGGACCGTG CTGACCCCCC TGCCATGGTG TGATCCGTAA AA -             #TAGGCACC     60                                                                 - - ATCAAAACGC AGAGG              - #                  - #                      - #    75                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:22:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                              - - GGAAGACGGG ATCGTTGC             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:23:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 51 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                              - - ACTCGGGCAA TTCGCCAAAC CGCAAGAACA GGCTGGCTGA CGTGGCTGGC G - #                 51                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO:24:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 51 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                              - - ATTCTTGCCG TCACGGCGCC CGTGACTTCG CCGGCATGGG CCACCCCGCA G - #                 51                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO:25:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 59 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                              - - AACATCACCG ACCTGTGCGC CGAATACCAC AACACCCAGA TCCATACCCT GA -             #ACGACAA      59                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:26:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 72 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                              - - GATCTTGTCG TTCAGGGTAT GGATCTGGGT GTTGTGGTAT TCGGCGCACA GG -            #TCGGTGAT     60                                                                 - - GTTCTGCGGG GT              - #                  - #                      - #       72                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:27:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 50 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                              - - GGCCCATGCC GGCGAAGTCA CGGGCGCCTG ACGGCAAGAA TCGCCAAGCC  - #                  50                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO:28:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 52 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                              - - ACGTCAGCCA GCCTGTTCTT GCGGTTTGGC GAATTGCCCG AGTGCAACGC AT - #                 52                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:29:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                              - - CCCGTCTTCC CCTCTGCG             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:30:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                              - - TTTTGATGGT GCCTATTTTA CGGATCACAC CATGGCAGGG GGGTCAGCAC GG -             #TCCGGACG     60                                                                 - - GTGACCGGTA C               - #                  - #                      - #       71                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:31:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 75 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                              - - CGGTCACCGT CCGGACCGTG CTGACCCCCC TGCCATGGTG TGATCCGTAA AA -             #TAGGCACC     60                                                                 - - ATCAAAACGC AGAGG              - #                  - #                      - #    75                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:32:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                              - - GGAAGACGGG ATGAACATG             - #                  - #                      - # 19                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:33:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                              - - TCTCTGTCAC GCATTGTCAA GGCGGCGCCC CTGCGCCGCA C    - #                      - #   41                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:34:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 61 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                              - - CACGCTGGCC ATGGCGCTGG GCGCGCTGGG CGCCGCCCCG GCGGCGCATG CC -             #ACCCCGCA     60                                                                 - - G                  - #                  - #                  - #                   61                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:35:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 59 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                              - - AACATCACCG ACCTGTGCGC CGAATACCAC AACACCCAGA TCCATACCCT GA -             #ACGACAA      59                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:36:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 72 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                              - - GATCTTGTCG TTCAGGGTAT GGATCTGGGT GTTGTGGTAT TCGGCGCACA GG -            #TCGGTGAT     60                                                                 - - GTTCTGCGGG GT              - #                  - #                      - #       72                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:37:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 61 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                              - - GGCATGCGCC GCCGGGGCGG CGCCCAGCGC GCCCAGCGCC ATGGCCAGCG TG -             #GTGCGGCG     60                                                                 - - C                  - #                  - #                  - #                   61                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:38:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                              - - AGGGGCGCCG CCTTGACAAT GCGTGACAGA GACATGTTCA T    - #                      - #   41                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:39:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                              - - CCCGTCTTCC CCTCTGCG             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:40:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                              - - TTTTGATGGT GCCTATTTTA CGGATCACAC CATGGCAGGG GGGTCAGCAC GG -             #TCCGGACG     60                                                                 - - GTGACCGGTA C               - #                  - #                      - #       71                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:41:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                              - - ATTCTGCCGA TTACTTCACT TCGCTGGTCG GAATATGCTT GC    - #                      - #  42                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:42:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 51 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                              - - ACTCGGGCAA TTCGCCAAAC CGCAAGAACA GGCTGGCTGA CGTGGCTGGC G - #                 51                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO:43:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 51 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                              - - ATTCTTGCCG TCACGGCGCC CGTGACTTCG CCGGCATGGG CCACCCCGCA G - #                 51                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO:44:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 59 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                              - - AACATCACCG ACCTGTGCGC CGAATACCAC AACACCCAGA TCCATACCCT GA -             #ACGACAA      59                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:45:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 72 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                              - - GATCTTGTCG TTCAGGGTAT GGATCTGGGT GTTGTGGTAT TCGGCGCACA GG -            #TCGGTGAT     60                                                                 - - GTTCTGCGGG GT              - #                  - #                      - #       72                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:46:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 50 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                              - - GGCCCATGCC GGCGAAGTCA CGGGCGCCGT GACGGCAAGA ATCGCCAGCC  - #                  50                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO:47:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 52 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                              - - ACGTCAGCCA GCCTGTTCTT GCGGTTTGGC GAATTGCCCG AGTGCAACGC AT - #                 52                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:48:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                              - - ATTCCGACCA GCGAAGTGAA GTAATCGGCA G        - #                  - #              31                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:49:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                              - - ATTCTGCCGA TTACTTCACT TCGCTGGTCG GAATATGAAC ATG    - #                      - # 43                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:50:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                              - - TCTCTGTCAC GCATTGTCAA GGCGGCGCCC CTGCGCCGCA C    - #                      - #   41                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:51:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 61 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                              - - CACGCTGGCC ATGGCGCTGG GCGCGCTGGG CGCCGCCCCG GCGGCGCATG CC -             #ACCCCGCA     60                                                                 - - G                  - #                  - #                  - #                   61                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:52:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 59 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                              - - AACATCACCG ACCTGTGCGC CGAATACCAC AACACCCAGA TCCATACCCT GA -             #ACGACAA      59                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:53:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 72 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                              - - GATCTTGTCG TTCAGGGTAT GGATCTGGGT GTTGTGGTAT TCGGCGCACA GG -            #TCGGTGAT     60                                                                 - - GTTCTGCGGG GT              - #                  - #                      - #       72                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:54:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 61 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                              - - GGCATGCGCC GCCGGGGCGG CGCCCAGCGC GCCCAGCGCC ATGGCCAGCG TG -             #GTGCGGCG     60                                                                 - - C                  - #                  - #                  - #                   61                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:55:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                              - - AGGGGCGCCG CCTTGACAAT GCGTGACAGA GACATGTTCA T    - #                      - #   41                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:56:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:                              - - ATTCCGACCA GCGAAGTGAA GTAATCGGCA G        - #                  - #              31                                                                    __________________________________________________________________________

What we claim is:
 1. A nucleic acid molecule, comprising a Bordetellapromoter selected from the group consisting of the tox, prn and fhapromoter operatively coupled to a heterologous gene encoding anon-Bordetella gene product, wherein the heterologous gene istranscriptionally regulated by the Bordetella promoter.
 2. The nucleicacid molecule of claim 1 wherein the non-Bordetella gene product isselected from the group consisting of an enzyme, an antigen, animmunogen, an allergen, an enzyme inhibitor, a hormone, a lymphokine, animmunoglobulin or fragment thereof, a toxin, a toxin subunit, amammalian protein, a structural protein and a receptor.
 3. The nucleicacid molecule of claim 1 wherein the non-Bordetella gene product is acholera toxin molecule.
 4. The nucleic acid molecule of claim 3 whereinthe cholera toxin molecule is the B subunit thereof.
 5. A recombinantstrain of Bordetella containing the nucleic acid molecule of claim 1 andexpressing said non-Bordetella gene product.
 6. A nucleic acid molecule,comprising a Bordetella promoter selected from the group consisting ofthe tox, prn and fha promoter operatively coupled to a heterologous geneencoding a non-Bordetella gene product and a nucleic acid sequenceencoding a leader sequence for secretion of the non-Bordetella geneproduct, wherein the heterologous gene is transcriptionally regulated bythe Bordetella promoter.
 7. The nucleic acid molecule of claim 6 whereinthe leader sequence is a leader sequence of a Bordetella protein orsubunit thereof or a fragment or analog of the Bordetella protein leadersequence retaining secretion-mediating properties.
 8. The nucleic acidmolecule of claim 7 wherein the leader sequence is selected from thegroup consisting of the Bordetella pertactin leader sequence and apertussis toxin subunit leader sequence.
 9. The nucleic acid molecule ofclaim 7 wherein the heterologous gene encodes a cholera toxin molecule.10. The nucleic acid molecule of claim 9 wherein the cholera toxinmolecule is the B subunit thereof.
 11. The nucleic acid molecule ofclaim 6 wherein the leader sequence is a leader sequence of anon-Bordetella protein or subunit thereof or a fragment or analog of thenon-Bordetella protein leader sequence retaining secretion-mediatingproperties.
 12. The nucleic acid molecule of claim 11 wherein thenon-Bordetella gene product is a secreted gene product and thenon-Bordetella leader sequence is the native leader sequence of saidsecreted gene product.
 13. The nucleic acid molecule of claim 12 whereinthe secreted gene product is a cholera toxin molecule.
 14. The nucleicacid molecule of claim 13 wherein the cholera toxin molecule is the Bsubunit thereof.
 15. The nucleic acid molecule of claim 6 wherein saidBordetella promoter is the fha promoter of Bordetella pertussis and theleader sequence for secretion of the non-Bordetella gene product is theleader sequence of the PRN protein.
 16. A nucleic acid molecule selectedfrom the group consisting of toxp/CTB-L/ctb, fhap/CTB-L/ctb,toxp/S1-L/ctb, toxp/PRN-L/ctb, fhap/S1-L/ctb and fhap/PRN-L/ctbwherein:toxp is the tox promoter of Bordetella pertussis, fhap is thefha promoter of Bordetella pertussis, CTB-L is a nucleic acid sequenceencoding a leader sequence for cholera toxin subunit, S1-L is a nucleicacid sequence encoding a leader sequence for Bordetella pertussis toxinS1 subunit, PRN-L is a nucleic acid sequence encoding a leader sequencefor pertactin, and ctb is a heterologous gene encoding a cholera toxinB-subunit.
 17. The nucleic acid molecule of claim 16 which isfhap/PRN-L/ctb.
 18. A nucleic acid molecule comprising a Bordetellapromoter coupled to a nucleic acid sequence encoding a non-Bordetellaleader sequence for secretion of a gene product, operatively coupled toacid leader sequence.
 19. The nucleic acid molecule of claim 18 whereinthe non-Bordetella leader sequence is selected from bacterial leadersequences, eukaryotic leader sequences and viral leader sequences. 20.The nucleic acid molecule of claim 19 wherein the non-Bordetella leadersequence is the leader sequence for the cholera toxin B subunit.
 21. Arecombinant strain of Bordetella containing the nucleic acid molecule ofclaim 18 and secreting said gene product.
 22. A method of expression ofa gene product, which comprises culturing a recombinant strain of claim21, and isolating the expressed gene product.
 23. A nucleic acidmolecule comprising:a first DNA sequence corresponding to a 5' flankingsequence of a selected Bordetella gene and disposed at the 5' end of thenucleic acid molecule, a Bordetella promoter selected from the groupconsisting of the tox, prn and fha promoter operatively coupled to aheterologous gene encoding a non-Bordetella gene product, a nucleic acidsequence encoding a leader sequence for secretion of the non-Bordetellagene product, the heterologous gene being transcriptionally regulated bythe Bordetella promoter, and a second DNA sequence corresponding to a 3'flanking sequence of the selected Bordetella gene and disposed at the 3'end of the nucleic acid molecule, said first and second DNA sequencespermitting specific integration of the nucleic acid molecule into theBordetella genome at a locus corresponding to the selected Bordetellagene.
 24. The nucleic acid molecule of claim 23 wherein said Bordetellapromoter is the fha promoter of Bordetella pertussis and the leadersequence for secretion of the non-Bordetella gene product is the leadersequence of the PRN protein.
 25. The nucleic acid molecule of claim 24wherein said first and second DNA sequences correspond respectively to5' and 3' flanking sequences of the fha gene of Bordetella pertussis topermit specific utilization of the nucleic acid molecule into thepresence of Bordetella pertussis at the locus corresponding to the fhagene.
 26. A plasmid adapted for transformation of a Bordetella straincomprising the nucleic acid molecule of claim
 23. 27. A plasmid adaptedfor transformation of a Bordetella strain comprising the nucleic acid ofclaim
 25. 28. A recombinant strain of Bordetella having a nucleic acidmolecule integrated into the genome thereof at the locus of a selectedBordetella gene selected from the group consisting of the tox operon,prn gene and fha gene, said nucleic acid molecule comprising a 5'flanking sequence of said selected Bordetella gene, a Bordetellapromoter selected from the group consisting of the tox, prm and fhapromoters operatively coupled to a nucleic acid sequence encoding aleader sequence for secretion of a non-Bordetella gene product; aheterologous gene encoding a non-Bordetella gene product, wherein aheterologous gene is transcriptionally regulated by the Bordetellapromoter, and a 3' flanking sequence of said selected Bordetella gene,said recombinant strain secreting said non-Bordetella gene product. 29.The strain of claim 28 wherein the Bordetella strain is a selected fromthe group consisting of a B. pertussis strain, a B. parapertussisstrain, a B. bronchiseptica strain and a B. avium strain.
 30. The strainof claim 29 wherein the Bordetella strain is a strain of B. pertussis.31. A method of expression of a non-Bordetella gene product, whichcomprises culturing a recombinant strain of claim 28, and isolating theexpressed non-Bordetella gene product.